Hematopoiesis depends on the dual ability of the hematopoietic stem cell (HSC) to balance self-renewal and proliferation while replenishing progenitors to supply the body's needs over a lifetime. Stress brought on by conditions such as infection, anemia or toxic chemotherapy can disrupt this critical balance resulting in bone marrow failure and ultimately death. Understanding the pathways controlling self-renewal and proliferation will provide pharmacologic opportunities to protect vital HSC function. Ott1, a gene isolated as a fusion partner in t(1;22) acute megakaryocytic leukemia, is essential for maintaining self-renewal of HSCs during proliferative stress. Ott1 is a spliceosome component, has a transcriptional activation/repression domain, RNA Recognition Motifs and possesses binding sites for Histone Deacetylase (Hdac), Notch-effector Rbpj and the histone methyltransferase Set1db. Although Ott1 has been found to interact with viral RNAs, no physiologic targets have been identified. Preliminary data shows Ott1 interacts with a region of the c-Mpl gene involved in alternative splicing. C-Mpl is the receptor for Thrombopoietin (Thpo), which is critical for megakaryocyte development and maintaining both quiescence and proliferative function in HSCs during stress. Low level c- Mpl responses support HSC quiescence and high signaling responses allow proliferation, yet the modulating mechanism is not known. Analysis of HSCs from conditionally-deleted Ott1 mice show a dramatic increase in the ratio of Mpl-TR isoform to Mpl-FL (full length). Mpl-TR has described dominant negative function in vitro and impairs HSC engraftment in vivo. Ott1 binds in complex to the alternatively spliced region on c-Mpl RNA. In addition, Ott1-dependent epigenetic modifications to the alternatively spliced region of c-Mpl, including histone deacetylation and H3K4me3 marking provide a potential mechanism capable of regulating the Mpl-TR:Mpl-FL ratio and thereby modulating response to Thpo. Activation of Notch also favors Mpl-TR production which through Ott1 interaction with Rbpj, may explain how the bone marrow niche regulates HSC quiescence and proliferation during stress. The molecular mechanism Ott1-mediated underlying c-Mpl alternative splicing will be investigated and the role of associated epigenetic modifiers established. The link between Ott1, Rbpj, and c-Mpl will be examined as a mechanism for Notch to control HSC Thpo response. Finally, the effects of Mpl-TR on HSC proliferation and quiescence in relation to withstanding hematopoietic stress will be determined. The comprehensive strategy utilizing genetic complementation, chemical inhibitors, and antisense oligonucleotides to target the pathways regulating c-Mpl alternative splicing in this proposal may identify novel pharmacologic approaches to address human disease arising from impaired HSC function such as recovery from cytotoxic chemotherapy, graft failure after stem cell transplantation and bone marrow failure syndromes such as aplastic anemia.